Acute myeloid leukemia (AML) is the most common type of acute leukemia among adults with overall poor prognosis. More specific therapies have been developed against FLT3(ITD/TKD)-positive AML (FLT3 tyrosine kinase inhibitors, TKi). Still, complete remissions are rare, and after initial response the leukemia invariably progresses within a few months. AML cells accumulate lethal DNA double-strand breaks (DSBs), but DSB repair pathways maintain their survival. BRCA1/2-mediated homologous recombination (HR) usually plays a major role in DSB repair. However, we reported that leukemias expressing AML1-ETO and PML-RAR, 15- 35% of karyotypic normal AMLs, and TKi ? treated FLT3(ITD/TKD)-positive AMLs (39% of karyotypic normal AMLs) are HR-deficient. Proliferation of HR-deficient solid tumor cells depends on DNA polymerase theta (Pol?) encoded by POLQ gene, which promotes a backup DSB repair called microhomology-mediated end-joining (MMEJ). According to cBioPortal database AMLs express the highest levels of POLQ mRNA among all tumors. Western blot analysis detected high levels of Pol? in AML cells when compared to normal counterparts. Altogether, these findings supported by preliminary data suggest that survival of AML cells (especially HR-deficient), which are under constant genotoxic stress, depends on Pol?. Therefore, Pol? may be a novel and promising drug target in AML. Our preliminary data suggest that AML-inducing mutations [FLT3(ITD/TKD) +/- TET2mut, DNMT3Amut, MLL-AF9, AML1-ETO] can modulate the sensitivity of AML cells to the inhibition of Pol?. Therefore, in Aim #1 we will inhibit Pol? (shRNA, mutants and Polq-/- cells) and introduce AML-inducing mutations (retroviral infection, CRISPR/Cas9) to pinpoint mutations, which predispose leukemia cells to be sensitive to Pol? inhibition +/- standard cytotoxic drugs. Using high throughput screening followed by biochemical testing and in vitro cellular sensitivity assays, we identified Pol? inhibitors (Pol?i) that specifically kill HR-deficient primary AML cells. In Aim #2 we will further develop Pol?i towards novel drug-like entities as targeted therapeutics for AMLs. In Aim #3 we will employ murine AML-like models, primary AML cells and xenografts to test therapeutic potential of inhibition of Pol? combined with (1) other DNA repair inhibitors (RAD52i, PARPi) against HR-deficient AMLs, and (2) standard cytotoxic drugs against HR-proficient AMLs. We anticipate that Pol?i developed in these studies will be effective against a wide-range of AMLs and ultimately will be advanced to clinical trials.